About thirty years ago, Sluyterman et al pioneered chromatofocusing, a new liquid chromatography method. It consists of descending retained pH gradients created by complex interactions between multi-component mobile phase buffers (polybuffers) and weak anionic stationary phases. Separation of charged macromolecules by differential binding to charged stationary phases by titration of the charges on the macromolecular surface as the result of varying pH resolves molecular species much better than shielding of binding to charged stationary phases by increasing salt concentration. Thus, it was hoped that chromatofocusing would widely supplant traditional salt elution, but that has not occurred. In large measure this is because chromatofocusing was discovered to have several serious shortcomings. In chromatofocusing, for each pH range over which one wishes to create a gradient, one has to create a separate buffer solution. For any given pH range over which the gradient is formed, the slope of the gradient in the column is proportional to that pH range, thus precluding controlled variations of the slope during repetitive formation of gradients having the same range. No single formulation of the polybuffers could maintain good buffering capacity over more than 3 pH units, necessitating the commercialization of three different formulations by the manufacturer, Pharmacia, to cover the pH ranges from 11 to 8, 9 to 6, and 7 to 4. The polybuffers are composed of a complex mix of small polyelectrolytes, some of which frequently bind strongly to proteins targeted for isolation, and are very expensive. Thus they are essentially never used in preparative protein purification. Despite these shortcomings, the methodology has found a renewed market position as the first dimension of the Beckman-Coulter ProteomeLab™ PF 2D Protein Fractionation System. This has come about because, despite the difficulties outlined above, uncontrolled retained pH gradients are still significantly better at fractionating proteins from complex mixtures than isocratic pH salt gradients are.
In the intervening years since Sluyterman et al's initial work, numerous attempts have been made to address chromatofocusing's shortcomings. Virtually all of them have consisted of relatively simple buffer combinations designed to interact with weak ion exchange stationary phases to create retained pH gradients on the stationary phase. In a few instances the reference buffers have been mixed externally, but no successful method to control the pH by purely external manipulations has been forthcoming. Generally, those attempting to improve on chromatofocusing have made the assumption that interactions between the buffers and the stationary phase that lead to complex and hard to control changes in eluant pH are inevitable and thus purely externally determined pH gradients are not practically achievable. The result has been the absence of patented marketable systems for producing fully controllable pH liquid chromatography gradients over wide pH ranges. Thus, a number of important problems remain unsolved: 1) software driven creation of multi-step pH gradients with a range of both positive and negative slopes limited only by the mixing accuracy of modern liquid chromatography systems; 2) the software driven ability to vary the slope of a pH gradient arbitrarily throughout a chromatographic separation independent of the initial and final pH and without changing the buffer chemistry with each slope change; 3) creation of controlled pH gradients on both anionic and cationic stationary phases; 4) software driven control of gradients of additional constituents (additives) to the eluting buffer such as nonionic detergents, organic solvents, salts etc. that do not significantly interfere with the formation of the pH gradients such that two or more independent, simultaneous gradients can be developed and controlled on the same stationary phase (s); 5) relatively inexpensive simple mobile-phase compositions with few buffer components while also solving problems #1-4; 6) buffer components of the mobile-phase that do not bind to proteins while also solving problems #1-5; and 6) provide mobile eluant phases that have hydrophobic, polar, and electrostatic properties allowing formation of efficient and independent simultaneous controllable gradients to fully utilize the selective and resolving capabilities of mixed mode stationary phases containing simultaneously ionizable groups and hydrophobic groups and stable over a broad pH range e.g. 2-12.